Endothelial Cell Tyrosine Kinase Receptor and G Protein–Coupled Receptor Activation Involves Distinct Protein Kinase C Isoforms

Protein kinase C (PKC) is a family of serine/threonine protein kinase isoforms that is important to intracellular enzymes for both tyrosine kinase receptors and G protein–coupled receptors. However, which isoforms are linked to which class of receptors in endothelial cell signaling is not known. Moreover, the PKC isoforms in endothelial cells have not been thoroughly characterized. We tested the hypothesis that specific PKC isoforms are involved in different signaling pathways. PKC isoform expression was assessed by using reverse transcription polymerase chain reaction and Western blotting. The spatial distribution of PKC after stimulation of the cells with basic fibroblast growth factor (bFGF) and thrombin was examined by using confocal microscopy. Expression of PKC α, δ, ε, θ, and ζ was detectable on both the mRNA and protein levels. In resting cells, PKC δ and ζ were mostly distributed in the cytosol, while PKC α and ε were also present in the nucleus. Nuclear immunoreactivity of PKC α and ε increased significantly between passages 1 and 3. The phorbol ester TPA induced a rearrangement of PKC δ and a translocation of PKC α and ε to the nucleus. Treatment of endothelial cells with TPA for 24 hours caused PKC α, δ, and ε to disappear, while PKC ζ was not influenced by TPA. bFGF induced a rapid assembly of PKC α along cytosolic structures, followed by a translocation of the isoform toward the perinuclear region and into the nucleus. bFGF had a similar effect on PKC ε. In contrast, thrombin had a smaller effect on nuclear translocation of PKC α, did not influence PKC ε, and induced a rapid nuclear translocation of PKC ζ. Thus, tyrosine kinase receptor activation via bFGF induced a rapid association of PKC α and ε with nuclear structures, while activation of the G protein–coupled thrombin receptor increased mostly nuclear PKC ζ. The translocation of PKC isoforms into the nucleus by growth-promoting factors may be important for the induction of endothelial cell growth.